Methods for diagnosis of peripheral nerve damage

ABSTRACT

Methods of diagnosing peripheral nerve damage, including diagnosing and monitoring chronic back and cervical pain are disclosed. The methods involve subjecting a body fluid sample from a patient suspected of having chronic lumbar or cervical pain and peripheral nerve damage to two-dimensional electrophoresis or an immunoassay and measuring relative amounts of protein or proteins which increase or decrease in concentration as compared to a standard control. A preferred method employs an Apo-E variant as a marker of peripheral nerve damage. Also disclosed are kits for use with the diagnostic methods.

This application is a divisional of Ser. No. 07/938,443, filed on Dec.1, 1992, now U.S. Pat. No. 5,364,793, which is a continuation-in-part ofPCT application Ser. No. PCT/US91/08552, filed on Nov. 15, 1991, whichis a continuation-in-part of U.S. Ser. No. 07/620,104, filed Nov. 30,1990, now abandoned.

FIELD OF THE INVENTION

This invention relates to novel methods for the diagnosis of peripheralnerve damage, including that damage which causes back and neck pain,particularly chronic back and neck pain.

BACKGROUND OF THE INVENTION

Conditions which cause pain are obviously very prevalent in medicine.Very often, the cause of the pain is apparent. However, frequently, thephysiological cause of the pain is not known. It is, of course,important to the clinician to determine the cause of the pain, so thatproper treatment can be instituted. Prime examples of painful conditionswherein it is difficult to determine the cause of the pain are inpatients experiencing spinal pain (i.e, lumbar, thoracic and cervical),particularly lower back ache or neck pain, and more particularly chroniccases. It is crucial in these conditions to determine whether they arecaused by muscle or fibrous tissue injury, or are actually a result ofnerve damage. The proper determination of the etiology will guide theclinician in the proper form of treatment.

Eighty-five percent of the United States population, at one time oranother, seek medical consultation for back ache, particularly chronicback ache. Over 40 million people claim disability due to chronic backpain (or low back syndrome) and the medical costs alone to care for thisgroup is over 40 billion dollars [Aronoff, G. M., Evaluation andTreatment of Chronic Pain, Urban and Schwarzenberg, Baltimore (1985)].This does not include the enormous socio-economic loss, estimated to bein the trillions of dollars. In 1985, 2.7 million individuals receivedsocial security disability insurance at a overall cost of $ 18.9 billion[Social Security Administration, Report of the Commission on Evaluationof Pain, Washington D.C., Department of Health and Human Services(1986)].

Clinically, chronic pain, as opposed to acute pain, is continuous painwhich persists for six months or more. Pain has been defined as anunpleasant sensory and emotional experience associated with actual orpotential tissue damage, or described in terms of such damage[International Association for the Study of Pain, chaired by Mersky(1979)]. Vasudevan has noted that there are several aspects to pain:nociception (the perception of pain, a physical stimulus);interpretation of the stimuli as "painful"; and the evaluation of thepain as creating suffering. [Vasudevan et al., "Counseling the Patientwith Chronic Pain-The Role of the Physician", In Persistent Pain, KluwerAcademic Publishers, Boston (1988)].

In the present state of the art, there is no objective, accurate testfor spinal pain, particularly chronic lower back (or lumbar) pain(hereinafter referred to as "CLP" which stands for "chronic lumbarpain") and/or radiating pain, and chronic cervical (neck) pain(hereinafter referred to as "CCP" which stands for "chronic cervicalpain"). More specifically, there is no protein-based clinical test whichquantitatively detects the presence or absence of CLP or CCP, much lessone having the capability to quantitate or monitor the progression orregression of CLP or CCP. The existence of such a test would be ofinfinite value to the patient, the doctor, and the society which bearsthe cost-burden of this problem (i.e., insurance companies andgovernment health and social security departments). An objective testfor CCP or CLP would allow for the following:

1. Verify the presence or absence of the syndromes of CCP or CLP basedon an organic cause. Ideally, this test would be performed on the firstvisit so that a baseline could be established to take advantage of thequantitative aspects of the test. If no organic cause existed, accordingto the test, then it would not be necessary to proceed with the morecostly examinations (e.g., MRI's, CT scans, myelograms, discograms, bonescans, electromyelograms, and consultations) which are, in the presentstate of the art, required to rule out causes for this syndrome. If anorganic cause is determined to exist, then the routine work-up couldproceed with high expectations for success. At this point, if theroutine tests for CLP or CCP are negative and the protein-based analysisdisclosed herein is positive for back syndrome, then the treatingphysician would be justified in continuing to seek a correctable,organic cause.

2. Monitor the progress and effectiveness of treatment. All chronic backor cervical syndromes are treated conservatively, initially. The effectof this treatment could be assayed and one or more of the followingjudgments could be made: continue with an improving test; discontinuewith a worsening test; change treatment with a worsening test; recommendinitial or revision surgery only when the test was worsening or notshowing any improvement. Thus, an objective, biochemical test wouldincrease the efficiency of conservative patient management and eliminateany unnecessary surgery. It is foreseeable that the test disclosedherein would become the standard for assessment, wherein surgery wouldbe indicated only if the protein analysis indicated it.

3. Identify the point of maximal medical improvement. By periodicallyadministering the test during a course of treatment, the quantitativecharacteristic of the test would allow the physician to assess thedegree of the symptoms of peripheral nerve damage of backache and/orradiating pain (particularly radiculopathy, which is currently thoughtto be due to nerve root damage) (CLP) and CCP and assist him inidentifying the point where medical treatment should cease. At thispoint, treatment and rehabilitation efforts can stop and the physician,patient, and employer can feel comfortable with a recommendation toreturn to full-time work, limited work, settle claims, retirement, etc.Medical costs should be reduced while the efficacy of medical treatmentimproves.

4. Identify those patients who are suffering disability from the pain ofCLP or CCP from those who are not suffering from the pain of neck pain,backache and/or radiating pain. (Radiating pain is defined as pain thatis perceived in one or both buttocks and/or one or both lowerextremities. Radiating pain is currently divided into two categories:(1) referred pain which means pain that radiates into the buttock(s) andthighs and remains above the knee; and (2) radiculopathy which meanspain which radiates into the buttock(s), thigh and below the knee,sometimes to the foot. Referred pain may be due to muscles, fascia,etc., while radiculopathy is thought to be due to nerve root damage.)This will assist the proper authorities in placing those who qualify forfinancial assistance because of an objectively documented back paincondition in the appropriate social program, and to identify and removethose who do not medically qualify.

5. Aid the courts and others concerned with assessing correctly thecompensable damages of pain and suffering secondary to neck pain,backache and/or radiating pain.

An objective test for peripheral nerve damage, in general, would allowthe clinician to verify whether patients with peripheral nerve problemswith neurological symptoms (for example: carpal-tunnel syndrome;brachial plexus problems; thoracic outlet syndrome; peripheral nerveinjuries; peripheral nerve damage as a result of disease, ageing,congenital abnormalities, neoplasms; optic or auditory nerve damage dueto many conditions, etc.) suffer from nerve damage, which would dictatea particular course of therapy.

Clinical tests for CCP or CLP include inspection, palpitation andmanipulation. The vast majority of clinical tests depends upon thepatient reporting a painful or other type of response, and are thereforeunreliably subjective. Objective clinical tests in the current state ofthe art include reflex changes, spasm and properly performed straightleg raising tests, and may or may not aid in the diagnosis of lower backsyndrome. Moreover, they neither quantitate nor monitor the progressionof lower back syndrome.

Thermograms, psychological interviews (e.g., McGill and MMPI tests),polygraphs and instrumentation tests may also be used to assist in thediagnosis of CLP and CCP. However, none of these is completely accuratebecause they are also subjective and depend on the patient reporting thetype and degree of response sustained.

Laboratory tests such as X-rays, CT scans, MRI's, myelograms,discograms, EMG's and bone scans can only delineate the presence orabsence of possible pain-producing lesions which must then be correlatedwith the clinical findings of CLP or CCP. They do not detect thepresence or absence of CLP or CCP per se, nor in any way quantitatethem. Further, it is not uncommon to have false positive and falsenegative results with these tests (reported rates of error of about20-50%). All or any of these tests may be negative and the patient maycontinue to complain; on the contrary, all or any of these tests may bepositive and a patient may remain asymptomatic. Moreover, not only arethese tests expensive, some of these tests expose the patient tounnecessary radiation.

The capacity to obtain diagnostic information from proteins,particularly blood proteins, has progressed rapidly since the middle ofthe 19th century when it was believed that serum contained but a singleprotein, albumin. By 1887, Lewith had demonstrated, by saltprecipitation, that serum proteins could be separated into the albuminsand globulins. The ratio of albumin to globulin (A/G ratio) was shown tohave diagnostic value and is still in use today. With the introductionof electrophoretic separations, immuno-analytic techniques and enzymaticassays, the number of plasma proteins of diagnostic value has grownexponentially. The examination of specific blood proteins has proven tobe an invaluable diagnostic aid, as in the monitoring of creatinephosphatase levels in determining cardiac damage following a myocardialinfarct. The increased resolution and detection of plasma proteins withtwo-dimensional electrophoresis [O'Farrell, J. Biol. Chem., Vol. 250,pp. 4007-4021 (1975)] combined with silver-staining [Merril, Proc. Natl.Acad. Sci., USA, vol. 76, pp. 4335-4339(1979)] allows investigators anexamination of over one thousand proteins in human plasma andapproximately 300 proteins in human cerebrospinal fluid.

Anderson et al. [Proc. Natl. Acad. Sci., USA, Vol. 74, pp. 5421-5425(1977)] initiated the mapping and the identification of the plasmaproteins resolved by two-dimensional electrophoresis. The goal of thiswork was to use these proteins for screening genetic variants. By 1984,they were able to identify only 38 of 646 serumproteins visualized bytheir electrophoretic and staining systems [Anderson et al., PlasmaProteins, Vol. IV, pp. 221-269, Academic Press, New York (1984)].

It has been suggested that two-dimensional gel electrophoresis can beused to correlate the presence of a protein in serum or tissue, or anincrease in its amount, with various diseases [Tracy et al.,"Two-Dimensional Gel Electrophoresis: Methods and Potential Applicationsin the Clinical Laboratory", J. Clin. Lab. Autom.,Vol. 3, No. 4, p. 235(1983)]. It has also been noted that development of a protein "profile"for disease states may be useful in diagnosis [Tracy et al., supra at242].

However, the increase in resolution provided by two-dimensionalelectrophoretic techniques and the increased detection available withrecently developed staining methods has not yet resulted in widespreadclinical applications of this methodology. Thus, the diagnoses ofdisease states in general, and chronic back pain in particular, by wayof two-dimensional gel analysis is new, there being only one suchreported method. This method utilizes two-dimensional gel proteinanalysis of cerebrospinal fluid to distinguish Creutzfeldt-Jakob diseasefrom other causes of dementia (Harrington et al., U.S. Pat. No.4,892,814).

Harrington et al. [Clinical Chem., Vol. 31, pp. 722-726 (1985)] alsofound some proteins associated with Parkinson's disease andschizophrenia, which may or may not be of diagnostic value. Someproteins mapped and identified by two-dimensional electrophoresis ofplasma [Anderson et al., 1984, supra] and cerebrospinal fluid [Goldmanet al., Clin. Chem., Vol. 26, pp. 1317-1322 (1980)] have demonstrated tobe polymorphic and thus may provide for genetic and forensicapplications, but have not proven reliable as diagnostic markers forparticular diseases.

To overcome the aforementioned deficiencies in the art, the presentinventors have developed an objective, quantitative test for diagnosingperipheral nerve damage, particularly that which causes spinal pain andmore-particularly CLP or CCP. The test utilizes two-dimensionalelectrophoresis to analyze the increased or decreased concentrations ofcertain proteins in a body fluid sample from a patient as compared to anormal control. During the course of developing this test, the presentinventors discovered a protein marker, which is indicative of peripheralnerve damage.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide for an objective,diagnostic test for peripheral nerve damage, particularly that whichcauses chronic spinal pain, and more particularly chronic lower back andneck pain. As used herein, "peripheral nerve damage" refers to allperipheral nerve problems with neurological symptoms (for example:cubital or carpal-tunnel syndrome; brachial plexus problems; thoracicoutlet syndrome; peripheral nerve injuries; peripheral nerve damage as aresult of disease, ageing, congenital abnormalities, neoplasms; optic orauditory nerve damage due to many conditions; conditions involving theautonomic nerve system; etc.).

It is a further object of this invention to provide a method fordetermining the severity of the peripheral nerve damage, particularlythat which causes chronic spinal pain and more particularly CLP or CCP.

It is yet a further object of this invention to provide for a method ofdetermining the type (i.e.,conservative versus surgery) and theeffectiveness of a course of treatment for conditions resulting fromperipheral nerve damage, particularly that which causes spinal pain andmore particularly chronic lower back or cervical pain.

In the course of developing the foregoing objects, the present inventorshave also identified proteins associated with CCP and CLP according totheir migration on two-dimensional polyacrylamide gels. Thus, theso-identified proteins and products derivable therefrom (for example,antibodies) are also part of the present invention. As one skilled inthe art would recognize, a spot on a stained two-dimensional gel couldrepresent one or more proteins.

A number of proteins detectable by the methods of the present inventionhave been found to be altered in concentration and/or migration pattern.As would be expected in studies of this nature, some protein spots havebeen found to be of a higher diagnostic value than others when subjectedto statistical analysis.

According to an initial study of the present invention, chronic lowerback pain is accurately diagnosed by the detection of increased ordecreased levels of forty-four proteins in patients suspected ofsuffering from chronic back pain as compared with normal controls (i.e.,normal volunteers with no clinical evidence of chronic back pain). Inpatients with chronic back pain, 29 proteins are found to be increasedto levels at least three-fold as compared to normal controls, withstatistical significance. (Statistical significance is defined herein asa p value of less than or equal to 0.05 by the Student t test or the logStudent t test.) Of these 29 proteins, 13 are found to only occur inchronic back pain patients. On the other hand, patients with CLP exhibitdecreased levels of at least three-fold of 13 proteins as compared tocontrols. Seven of these proteins are totally absent in patients withthe back pain syndrome. A further investigative study ("second study")found one additional spot which has very high predictability of thepresence or absence of CCP and CLP and, thus, is favored for diagnosticvalue, as well as ease of observation.

Thus, the diagnostic methods of the present invention can employtechniques to identify the increase or decrease, or presence or absence,of these proteins in a sample to diagnose CLP or CCP. Of course, thepresence or absence of a defined protein or proteins in a spot in aspecific location on the gel may be due to changes in the migration ofproteins altered in charge or molecular weight. Also, one may performmultivariate analyses on an array of more than one of the proteins byusual statistical methods.

These proteins are identified by their relative molecular weight andisoelectric point (i.e., their migration on two-dimensionalelectrophoresis gels). The exact identity of only one of the proteins isknown (spot 1bp13-14.719). However, as CLP and CCP may be associatedwith inflammation, some of the proteins which appear to be increased inCLP and CCP may belong to the class of plasma proteins which are knownto be increased in response to tissue injury and disease. This class ofproteins was first discovered by the observation of their induction inpatients with pneumococcus pneumonia [MacCarty, M., "HistoricalPerspective on C-Reactive Protein.", In Kushner et al (Eds.,) C-ReactiveProtein and the Plasma Protein Response to Injury, Annals of the NewYork Academy of Sciences, Vol. 389, pp. 1-10 (1982)]. Since theseinitial observations, the metabolic and physiological changes that occurin the acute phase response have been studied in numerous laboratories.It has been found that the acute phase response may be invoked by manydifferent types of stimuli, such as trauma, infections, noninfectiousinflammatory states, and tissue infarctions. See, Kushner et al. (Eds.),supra. While it is known that most of these proteins are synthesized inthe liver, the nature of their induction is not yet known. Inductioncould be by blood borne substances or by neuronal factors since thereare both blood vessels and nerves in the region of synthesis, thehepatic lobes [MacIntyre et al., "Biosynthesis of C-Reactive Protein.",In Kushner et al. (Eds.), supra, pp. 76-87].

Some of the acute phase response proteins have been induced in mice, andtheir relative positions have been identified with two-dimensionalelectrophoresis [Pluschke et al., Clin. Exp. Immunology, Vol. 66, pp.331-339 (1986)]. The present applicants could not be sure that theproteins affected in the present invention are the previously observedacute phase response proteins or, perhaps, new members of this class ofresponse proteins. However, the applicants sent an aliquot from each ofthe clinical samples to a commercial laboratory for measurement ofcomplement C3, alpha-1 antitrypsin, transferrin, alpha-1 acidicglycoprotein, and C-reactive protein (some of the well characterizedacute phase response proteins). No elevation of these proteins could bedetected by standard assays.

One of the markers found in the present invention to be a highlypredictable marker of CLP or CCP is the spot referred to as 1bp13-14.719(or sometimes referred to herein as "719"). The present inventorsfocused on this particular marker, and further investigations revealedthat this spot is actually an apolipoprotein E variant. It is documentedin the art that apolipoproteins accumulate markedly in the areaimmediately local to the nerve tissue during the regeneration of damagedperipheral nerves (less in the regeneration of damage to the CNS). Thisled the present inventors to investigate whether peripheral nervedamage, in general, would show increased amounts of spot 719 in theplasma of patients with peripheral nerve damage other than that nervedamage which causes CLP and CCP. As disclosed in the presentapplication, positive results for the increase of the apo-E variant wereseen in patients with other types of peripheral nerve damage. It shouldalso be noted that increases in the density of spot 719 can be seen withthe naked eye on two-dimensional gels, without the need for computerscanning densitometry. When quantitative measurements of density aremeasured, this spot 719 is about five-fold (can range from 2 to 5-fold)greater in spot density in peripheral nerve damage of patients ascompared to normal controls.

Based on the data obtained by the present inventors in this applicationin connection with chronic conditions, and in view of the contemporaryknowledge of nerve damage in the literature, it is contemplated that themethods of the present invention can be used to diagnose peripheralnerve damage at any time after injury to the nerve has occurred (i.e.,in the acute phase as well as the chronic phase), particularly withrespect to the observation of spot 719 (the apo-E variant).

It is unlikely that the protein changes noted herein are due to drugs,such as those the patients may have taken to alleviate their pain, sincethree of the patients in the initial study were not taking anymedication for their chronic back pain. These three patients displayedprotein alterations that were similar to those taking medication. It isalso unlikely that the protein changes are artifacts of storage. Tracyet al. demonstrated the occurrence of plasma proteins which are alteredby freezing and storage at -20° C. [Tracy et al., Clin. Chem., Vol. 28,pp. 890-899 (1982)]. Our spots 1305, 1318, 1323 and 4614 are in theregion noted for the appearance of such spots by Tracy et al. However,as the patient and the age and sex matched control samples were drawn atthe same time and stored under identical conditions, it is unlikely thatthe proteins of interest in this study are storage artifacts.

The methods of the present invention include one of particularlysignificance. That is, the present inventors have developed a plasmatest for peripheral nerve damage and repair by focusing their attentionon the apo-E variant of spot 719. Secondarily, this discovery leads to aplasma test for spinal pain and other pain due to nerve damage.Consequently, the present inventors have discovered a plasma test forpain. In other words, peripheral nerve damage can produce spinal pain(and/or other neurological deficits) and pain (and/or other neurologicaldeficits) outside the spinal column. Peripheral nerve damage can bediagnosed by the presence of an increase in the apo-E variant (spot 719)in the plasma. Therefore, spinal pain (and/or other neurologicaldeficits) and pain (and/or other neurological deficits) outside thespinal column can be diagnosed by an increase in the plasma apo-Evariant.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Plasma gel stained with silver. This gel was made with 1.47 ulof plasma from a control. Circles mark proteins not generally visible incontrols but present in patients with chronic back pain. Numberspreceded by `M` designate landmarking proteins identified in Table I.The remaining labeled proteins are those which either increased ordecreased by a factor of three or more and were statisticallysignificant, as indicated in Tables II and III.

FIG. 2: A computer generated diagram of all the proteins analyzed in theinitial study. The numbered proteins are the same as those identified inFIG. 1.

FIG. 3: A scatter diagram illustrating proteins which showed robustcorrelations with CLP in the initial study. The numbers to the left ofthe circles indicate the frequency greater than 1 that a patient withthat value was observed. The circle and bar to the right of each groupof data indicate the mean and the standard error of the mean for thatgroup.

FIG. 4: A scatter diagram illustrating proteins which showed moderatelyrobust correlations with chronic back pain in the initial study. Thenumbers to the left of a circle indicate the frequency greater than 1that a patient with that value was observed. The circle and bar to theright of each group of data indicate the mean and the standard error ofthe mean for that group.

FIG. 5: A patient by patient comparison of protein 1318 densities withthe degree of lower back disability. The degree of disability was scoredby using a number of factors, such as: measurements of back and legmotion limitations, abnormalities in the knee jerk reflexes and historyof back surgery.

FIG. 6: A patient by patient comparison of protein 1316 densities withthe degree of lower back disability.

FIG. 7: A patient by patient comparison of protein 1204 densities withthe degree of lower back disability.

FIG. 8: A patient by patient comparison of protein 1305 densities withthe degree of lower back disability.

FIG. 9: A patient by patient comparison of protein 3203 densities withthe degree of lower back disability.

FIG. 10: A patient by patient comparison of protein 3211 densities withthe degree of lower back disability.

FIG. 11: A patient by patient comparison of the average of proteins 1316and 1318 densities with the degree of lower back disability.

FIGS. 12-14: 2-D gel images of three patients with chronic lower painback in the second study, showing the spot 1bp13-14.719.

FIGS. 15-17: 2-D gel images of three controls run side-by-side with thegels of FIGS. 12-14. Open circles represent area where 1bp13-14.719 spotis missing.

FIGS. 18-20: Represent enlargements of the areas blocked off in FIGS.12-14.

FIGS. 21-23: Represent enlargements of the areas blocked off in FIGS.15-17.

FIGS. 24a-c: These are photographs of 2-D gels of the present invention.FIG. 24a shows the location of spots other than spot 719 (such as otherapolipoproteins and some acute phase reactant proteins). See further thelegend on FIG. 25a for description of spot numbers. FIG. 24b is a 2-Dgel of a patient with CLP; the boxed area is enlarged in the lower,right-hand corner of the figure. FIG. 24c is a 2-D gel of a normalcontrol showing a much-diminished, barely-visible spot 719; as with FIG.24b, the lower, right-hand corner of the figure is an enlarged view ofthe boxed area.

FIGS. 25a and b: This set of figures are bar graphs comparing themeasured density [% TID (or total integrated density)] of the spotsshown in FIGS. 24a-c between chronic lower back pain patients and normalcontrols. While there are observed increases in the otherapolipoproteins, these have not been proven to be as statisticallysignificant as spot 719 (apo-E variant).

FIG. 26: Immunoblot prepared in accordance with the example in thepresent invention, showing spot 719 is positive for anti-apo-Ereactivity. The other positive spots (680 and 684), other forms ofapo-E, were also analyzed for quantitative variations correlating withCLP, but there was no significant correlation (see FIGS. 25a and b).

FIG. 27: N-terminal sequence analysis of spot 719 protein [SEQ ID NO:1].There is 100% homology with the known N-terminal sequence of plasmaapo-E [SEQ ID NO:2].

DETAILED DESCRIPTION OF THE INVENTION

This invention involves methods of diagnosing peripheral nerve damage,particularly that which causes spinal pain, and more particularly CLPand CCP wherein protein samples from both normal and abnormalindividuals are subject to electrophoresis and/or immunoassays. In thecase of two-dimensional gel electrophoresis, a large number of proteinspots common to both types of individuals and spots which appear ordisappear in the abnormal patient group are determined. Initially, thenumber of protein spots to be examined is reduced to only those showingstatistically significant differences between normal controls andpatients with chronic back pain. This is determined by performing aStudent's t test or a log Student's t test on the spot intensity data.Those proteins that have statistical differences at a significance levelof 0.05 on either or both of these tests are chosen for further study.In addition, the present invention also contemplates the use ofone-dimensional electrophoresis.

CLP has been shown to arise from trauma to spinal nerve roots[Schonstrom, N. et al., Spine, Vol.9, pp. 604-607 (1984)]. While it isdifficult to examine biochemical alterations in human nerve injuries,molecular changes associated with nerve root damage have been studied inseveral animal models. [Ignatius, M. J., Progress in Brain Research,Vol.71, pp.177-184 (1987)]. Changes in the injured nerve includeelevation in the local concentrations of acute phase reactant proteins,infiltration by circulating monocytes, increased levels of proteinsynthesis and increases in apolipoprotein concentrations. One of themost striking physiological differences in these animal models of nervedamage is a 250-fold increase in the local concentration ofapolipoprotein E (apo-E), with sciatic nerve crush injuries [Skene, J.H. P, Proc Natl. Acad. Sci. U.S.A., Vol. 80, pp. 4169-4173 (1983)]. Thisnerve damage-associated elevation in the area surrounding the nervecorrelates with the present inventors' finding that a subset of theapolipoprotein E complex of spots in our 2-D gels was increased greaterthan five-fold in the plasma of individuals suffering CLP and CCP. Otherapolipoproteins and acute phase reactants that might be expected to beelevated in plasma of individuals suffering nerve root damage, based onthe animal studies, (most notably apo-D, apo-AI and apo-AIV) wereidentified by their 2-D gel location, but were found not to be elevatedto a statistically significant degree in our analysis. To betterunderstand the appearance of the apolipoprotein E variant (spot 719),the present inventors wish to determine the following: (1) was painrequired for the protein change or was only a biomechanical abnormalitynecessary?; (2) was it limited to the lumbar region or did it apply tothe whole spine?; (3) was it present in all individuals with acute orchronic pain?; (4) was it found in cases of peripheral nerve damage?;and (5) was it associated with an inflammatory response? To help answerthese questions, the present inventors also analyzed the plasma ofpatients suffering from a variety of other chronic and acute painfulconditions, as well as patients that had recovered from CLP andindividuals with chronic inflammatory conditions. Based on this analysisand previously-reported observations, the present inventors proposedthat the apo-E abnormality results from a chronic inflammatory insult,causing continuous attempts to regenerate damaged nerve. Further,although other investigators have documented that the concentration ofapo-E immediately surrounding the damaged nerve tissue increasesgreatly, none of the prior art discloses or suggests that an increase inapo-E in the plasma, in general, and human plasma, in particular, wouldalso occur.

In order to better understand the reason for increased concentration ofspot 719 in the present studies, the present inventors undertook toidentify the protein comprising this spot. Two-dimensional gel maps ofplasma proteins were used to locate spot 719 in the area of thetwo-dimensional gel, which contains transthyretin ("TTR") dimer and theapo-E complex of spots. Spot 719 appears to have a relative molecularweight of about 32-36 kD and a pI of about 6.0-6.2 as determined fromthe 2-D gels. Immunoblot analysis of the two-dimensional gel showedstrong reactivity of spot 719 within the apo-E monoclonal antibody (FIG.26). N-terminal microsequence analysis was performed to confirm theidentity of spot 719 as apo-E (FIG. 27). In addition, microsequenceanalysis independently verified the identity of another spot in theregion not related to lower back pain as TTR. Circulating plasma apo-Eis active in lipid transport from the gastrointestinal system to theperiphery through the plasma. In addition to this endocrine function,apo-E also has paracrine and autocrine activities for cholesterolredistribution. It has also been shown to be a regulator of the immuneresponse and a neurotropic factor. The paracrine activity of apo-E innerve regeneration has been well described in sciatic nerve crush inrats [Schubert, D. et al., J. Cell Biol., Vol. 104, pp.635-642 (1987)].In experimental models, by three weeks after nerve injury as much asfive percent of the total soluble protein of the nerve is apo-E [Mahley,R. W. et al., Science, Vol. 240, pp.622-630 (1988)]. While in the CNS,there are similar levels of apo-E synthesis in response to injury,response to accumulation around the tissue is not observed [Muller, H.W. et al, Science, Vol. 228, pp. 499-501 (1985)]. It has been suggestedthat the lack of accumulation of apo-E in the CNS is an important factorin the lack of regeneration of neurons in the brain.

The source of the apo-E comprising spot no. 719 is not obvious. Theblood nerve barrier in the dorsal root ganglion and other nerve rootshas been shown to be compromised by nerve crush injury. [Howe, J. F. etal, Pain, Vol. 3, pp. 25-41 (1977); and Wiesel, S. W. et al, Spine, Vol.10, pp. 549-551 (1984)]. This may lead to proteins entering thecirculation at the point of injury, although this is mere speculation.The extraordinary amount of paracrine apo-E that can enter thecirculation at the site of injury may account for the observed increaseof this polypeptide in the plasma. However, it is very noteworthy thatelevated levels of the other apolipoproteins observed in the literature(e.g., apo-D, apo-AI and apo-AIV) to accumulate in the local area aroundthe nerve tissue do not correlate with statistically significantincreases in levels in the plasma, as the present inventors haveobserved with the apo-E of spot 719.

It is also possible that the source of increased apo-E in the plasma ofindividuals with CLP or CCP is endocrine and produced in response toacute phase reactants resulting from the injury. However, the levels ofthe few acute phase reactants whose locations can be determined on 2-Dgels do not differ between CLP patients and normal controls (see FIG.25a and 25b). While the source of the apo-E/spot 719 increase has notyet been identified, the present inventors believe its variation inindividuals suffering CLP and CCP is important in the diagnosis andmanagement of these conditions, as well as in peripheral nerve damagegenerally. It may also provide insight into the mechanism of neuronalregeneration.

Any number of protocols can be used to develop protein data for use inperforming the diagnostic methods of the present invention. The protocolused in the present studies and as exemplified herein was approved bythe IRB of St. Luke's Hospital in Houston, Tex., and patients and sexand age matched volunteers each of whom signed an informed consentletter. The patients were complaining of chronic (six months or more induration) low back pain secondary to a reported injury and were randomlyselected from one of the applicants orthopedic practice. The patientswere requested to remain drug-free for at least one week prior to bloodsampling. The controls were free of significant medical problems asdetermined by medical history and physical examination. The initialstudy consisted of 10 patients and 10 sex and age matched controls. Inthe second study, a similar protocol consisted of 64 plasma samples from36 lower back pain patients and 28 controls. The data for this studywere obtained from three separate studies: two independent blindedstudies, performed using the apo-E variant (spot 719) to determine whichindividuals had CLP, provided the correct diagnosis in sixteen out ofseventeen patients (94.1%) and 14 out of 14 controls (100%); and a thirdstudy, which was not blinded, was performed and was accurate in 18 outof 19 patients (94.7%) and 14 out of 14 controls (100%).

Additional studies were undertaken which focused on spot 719. Thesestudies are set forth in the examples which follow.

To prevent degradation of samples of tissue, serum, or other body fluids(preferably blood and more preferably plasma) from the subjects, thesample is initially frozen in dry ice. At any point prior toelectrophoresis a portion of the sample may be removed for counting andassaying the amount of protein by, for example, the Lowry method.

The first stage gels for the two-dimensional electrophoresis generallycontain urea at a concentration of about 9M and about 2% nonionicdetergent, both of which aid in dissociating proteins. The nonionicdetergent helps keep the separated proteins from precipitating at theirisoelectric points. These reagents and their proportions can varysomewhat, provided that these objectives are accomplished. An ampholyte(e.g., 2% 4-8 pH ampholyte) is also desirable to maintain a pH gradientacross the length of the gel, although other reagents which maintain thepH gradient could be substituted. The acrylamide concentration of thefirst stage gel should be such as to permit protein movement to theisoelectric point. The first stage gel can be in a number of forms; forexample, it can be housed in an isoelectric focusing tube or in a slabform. Preferably, the first dimension is in the form of a tube gel.

The samples are usually prepared for the first stage by solubilizing ineither 10% sodium dodecyl sulfate (SDS) or urea at a concentration ofabout 9M. The reducing agent 2-mercaptoethanol is also usually includedto separate disulfide-linked subunits. An ampholyte to maintain the pHgradient, a nonionic detergent which does not affect the protein charge,and dithiothrietol (DTT) which disrupts disulfide bonds, may also beincluded. Other reagents may also be added, or other reagents whichaccomplish the foregoing functions may be substituted. For example,prior to subjecting the samples to gel electrophoresis, the samples maybe placed in a sample buffer (for example, 2% SDS, 2% DTT, 20% glycerol,2% ampholines and 2% CHAPS) and placed in a boiling water bath (at about100° C.) for about two minutes to aid in dissolution. This temperatureand the time exposed thereto has been found to not cause proteindegradation; however, both can be varied provided that dissolution takesplace and protein degradation does not. The sample buffer unfolds theprotein, separates the disulfide-linked subunits, and maintains the pH.Other sample buffers which accomplish the foregoing functions, and othermethods of dissolving the protein samples, can also be used.

The samples may then be cooled on ice and treated with DNase and RNaseto reduce the viscosity. The samples can then be snap-frozen in liquidnitrogen and packed on dry ice if they are not to be run on gelsimmediately. This adequately preserves the samples. However, it has beenfound that any method that cools a dissolved sample to -70° C. or morewill also preserve the samples.

Polyacrylamide gel electrophoresis in the presence of SDS is usuallyused for the second dimension separation. SDS is an ionic detergent andbinds strongly to proteins. It eliminates the native protein chargecharacteristics and unfolds the protein into a rod-like form. See Tracyet al., J. Clin. Lab, Autom., supra. Thus, when protein is subjected toan electric field in the polyacrylamide gel matrix, the uniform negativecharge and the relatively uniform shape of the SDS-protein complexesallow separation essentially by molecular weight, with thepolyacrylamide gel matrix acting as a sieve.

The second stage gel is preferably SDS-equilibrated, to eliminate theprotein charge, and contains a higher acrylamide concentration than thefirst stage gel, to aid in separating proteins by molecular weight.Other reagents can be added or substituted. Further, the second gel canbe in a variety of forms; preferably, however, the second stage is inthe form of a slab gel.

The spots on the gels can be viewed by any number of methods includingstaining with Coomassie blue and silver staining. They can be visualizedfor relative protein density manually, but it is preferred that they bescanned with an appropriate camera system with a normalization standard(available from the National Bureau of Standards, Gaithersburg, Md.) andanalyzed with a computer densitometer to measure relative protein spotstaining intensities or densities.

In order to perform immunological tests for the diagnosis and/ormonitoring of CLP or CCP, the first step is to obtain antibodies to theproteins of interest. There are many methods of accomplishing this whichare well known to those skilled in the art. (For comprehensivelaboratory methods, see Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988), which is incorporated by referenceherein.) For antibodies with sufficient specificity for western blotsand immunoassays, the antigen must be purified to homogeneity or theantigen should be used to prepare monoclonal antibodies. Since theproteins of interest in this invention are seen as unique spots on thesecond dimension polyacrylamide gel, preferably the gel can be used asthe final purification step of the individual antigens. One can obtain apure antigen preparation by excising the spots which show increased ordecreased intensity in CLP or CCP patients. This gel piece can beinjected into an animal to raise antibodies. Alternatively, one may cutout the spot of protein and electroelute it from the gel to obtain aprotein in solution for injection. Still another technique forprocessing the protein for injection after separation on gels is toelectrophoretically transfer the proteins to nitrocellulose, locate theproteins of interest by staining (e.g., with Ponceau S), excise thespots and cut into pieces for injection. The particular method used isonly limited by the ability to elicit an immune response to the proteinsof interest.

As an alternative to using the antigen purified by separation on anelectrophoretic gel directly, one may elute a protein spot, obtain apartial sequence by any method well known in the art, and use thesequence to manufacture synthetic peptides (usually with an automatedmachine using solid-phase techniques). The synthetic peptide should beat least six amino acids long to elicit antibodies that bind to theoriginal protein. The purified synthetic peptides would then be coupledto carrier proteins, and these conjugates are then used to immunizeanimals.

The polyclonal antibodies in the antisera obtained with the foregoingmethods can be used for western blots and other immunological tests.However, one may further utilize hybridoma technology to obtainmonoclonal antibodies, which may be the best choice for immunochemicaltechniques. Methods of monoclonal production are well known in the artand were first described by Kohler and Milstein in 1975. Briefly,antibody-secreting cells are fused to, for example, myeloma cells tocreate hybridoma cells which are cloned and screened by appropriatemethods for the desired antibodies. While a monoclonal antibody forapo-E is available (Chemicon), this antibody reacts not only with spot719, but with the other apo-E spots on the 2-D gel. In addition, theinventors' investigations showed that this commercially availablemonoclonal antibody does not demonstrate quantitative differencesbetween CLP plasma and normal plasma. Therefore, it is an object of thepresent invention to obtain a monoclonal antibody specific for spot 719to overcome the disadvantages with the currently available apo-Emonoclonal. It is only spot 719, out of the complex of apo-E spots onthe 2-D gel, which is increased in patients with peripheral nervedamage, in particular those patients suffering from CLP or CCP. Thelocation of this spot on the 2-D gels suggests that it is a variant formof apo-E, which differs from the other apo-E proteins by itsglycosylation or phosphorylation pattern or other novelpost-translational event, or that it is associated with another proteinor lipid which is not completely denatured prior to gel analysis.

One of the diagnostic methods of the present invention involves thedetection of proteins which are present in patients with CLP or CCP, yetabsent in normal controls. Besides locating these protein spots bystaining on a two-dimensional gel, one may detect the proteins byimmunoblotting, or western blotting, utilizing the polyclonal ormonoclonal antibodies raised to the particular protein of interest.Protocols for immunoblotting are well known in the art and generallycomprise the steps of gel electrophoresis, transfer, blocking, additionof antibody, and detection. The preparation of the sample and thetwo-dimensional gel electrophoresis is discussed above. At thecompletion of electrophoresis, proteins are transferred from the gel toa matrix, such as nitrocellulose, activated (diazo groups) paper andactivated (positively charged) nylon. Nitrocellulose membranes arepreferred for relatively low background and cost considerations;however, any membrane which will sufficiently bind the transferredproteins can be used. Preferably, transfer of the proteins isaccomplished by electrophoretic elution; however, simple diffusion orvacuum-assisted solvent flow can also be used. After transfer, themembranes may, optionally, be stained to determine the position ofmolecular weight markers.

Prior to antigen detection, one must block the membrane to preventnon-specific adsorption of immunological reagents. Most preferably, theblocking solution would be composed of nonfat dried milk or bovine serumalbumin. After blocking, antigens can be detected directly orindirectly. Direct detection utilizes labelled primary antibodies. Theantibodies, labelled with iodine, enzymes or biotin, can be prepared bymethods well known to those skilled in the art. In indirect detection,the primary antibody (unlabelled) is first added to membrane, followedby a secondary antibody (an anti-primary antibody) which is labelledwith radioactive iodine or an enzyme, such as horseradish peroxidase.The antigen is then detected by exposing a radiolabeled membrane toX-ray film or, in the case of enzyme-labelled antibody, by addingsubstrate to the membrane.

Alternative method of quantifying or detecting the presence of proteinfor the diagnosis of CLP or CCP is the use of immunoassays performeddirectly on the body fluid sample. Several immunoassays would be usefulin the context of the present invention, including: antibody capture (Abexcess); antigen capture (antigen competition); and the two-antibodysandwich technique. All immunoassays rely on labeled antigens,antibodies, or secondary reagents for detection and quantitation. Thelabel used can be radioactive or enzymatic, or one may label withfluorochromes or biotin. The choice of label is a matter of discretionwith the diagnostician, taking into consideration cost, sensitivity,radioactivity exposure, etc. The term "label" as used herein refers toany of the foregoing.

In an antibody capture type of assay, the test sample is allowed to binddirectly to a solid phase and any unbound antigen is washed away. Theantibody specific for the antigen is added and allowed to bind. Theamount of antibody bound to the solid phase, after washing away unboundantibody, is determined using a secondary reagent. Suitable secondaryreagents include anti-immunoglobulin antibody, protein A or protein G.These can be obtained from commercial sources or prepared by methodsknown in the art. Detailed protocols can be found in Harlow et al.,supra, and are incorporated herein by reference, the particular methodsused not being limited or essential to the practice of the presentinvention.

Antigen capture type assays measure the amount of antigen in a testsample via a competition between labeled and unlabeled antigen. Thistype of assay is exemplified by a "radioimmunoassay" or RIA. The firststep in this type of assay is to bind unlabeled antibody to a solidsupport (either directly or through an intermediate protein, e.g., ananti-immunoglobulin antibody). A sample of known antigen of knownquantity is labeled and a sample of this is added to the test materialcontaining an unknown amount of antigen, and the mixture is added to thebound antibody. The antigen in the test sample competes with the labeledantigen for binding to the antibody bound to the solid support.Following removal of the unbound antigen, the amount of labeled boundantigen is measured. The higher the concentration of antigen in theunknown test sample is, the more effectively it competes with thelabeled antigen; therefore, a decreasing amount of label is detectedwith an increasing amount of unlabeled antigen. Thus, generated standardtitration curves will yield relative levels of antigen.

Another immunoassay to quantitate antigen concentration is thetwo-antibody sandwich technique. This type of assay requires twoantibodies that bind to two separate epitopes of the antigen. Thus, onemay use two monoclonals that recognize two separate sites on theantigen, or a batch of purified polyclonals can be used. The essentialsteps are as follows: 1) one purified antibody is bound to a solid phaseand the antigen in the test sample is allowed to bind to the firstantibody; 2) unbound antigen is washed away and a labeled secondantibody is allowed to bind to the antigen; and 3) after washing, thesecond labeled antibody that is bound to the matrix is quantitated. Asin other assays, a standard titration curve with known dilutions isplotted and the unknown sample is compared thereto. In order todetermine absolute amounts, a standard curve generated with knownquantities of antigen is used.

A wide variety of test kits are possible to take advantage of theadvances in the diagnostic arts made possible by this invention. Somewill be described here; others can be devised by those skilled in theart.

The central reaction in a test kit could be between any one of theaberrant proteins found in patients with peripheral nerve damage, inparticular that which causes CLP and CCP and the antibodies prepared asset forth above and in the Examples below. The Examples below aredirected to the preparation of antibody from rabbits, and the followingdescription and other Examples of test kits and test methods will bebased on the rabbit preparation. The rabbit is the preferred source ofimmunoglobulin and its fractions; however, the skilled artisan willrecognize that the following Examples utilize the rabbit only asexemplary. Other animals can be used and this will require somemodification of the other reagents used in the tests and the kits, andare readily apparent to one skilled in the art.

In the test kits, any of a variety of adsorbents can be used including,for example, glass or plastic surfaces which may be the inner surfacesof test tubes or the surfaces of test plates. Examples of flat surfacesespecially useful in an enzyme-linked immunosorbant assay (ELISA) or aradioimmunoassay (RIA) include glass, nitrocellulose paper, or plasticssuch as polystyrene, polycarbonate or various polyvinyls. The ligandscan be attached to the surface by direct adsorption, forced adsorptionand coupling, in accordance with known procedures. Typical test kits areset forth in the Examples below.

The following Examples illustrate the utility of the diagnostic methodsof the present invention, and are not intended to limit the scope ofthis invention. For instance, any of the known immunoassays or otherknown methods of protein detection may be used to aid in the diagnosisof peripheral nerve damage, in particular that nerve damage which causesCLP or CCP. Also, the method of diagnosis is not limited to the specificproteins elucidated by the present Examples. Modifications of theprocedures as would be apparent to one skilled in the art are within thescope of the teachings.

EXAMPLE 1

In a first (initial) study, ten patients with chronic back pain wererandomly selected from a group of patients complaining of CLP of sixmonths or more secondary to a reported injury. These patients, 3 femalesand 7 males, ranged in age between 20 and 55 years. Of these patients, 7were taking medication for their pain. However, 3 of these patients tookno medication. The degree of lower back disability was evaluated by anumber of factors such as: the history of the back pain (includingradiations and the induction of pain with coughing and/or sneezing);physical examination including the loss of sensitivity in thedermatomes; measurements of back and leg motion limitations;abnormalities in the ankle and/or knee jerk reflexes; and the analysisof spinal radiographies (for spondy losis, stenosis, herniated discs,degenerations, the narrowing of the intervertebral space, etc.) andother special studies such as MRI, CT scan, myelograms, discograms andelectromyelograms. Controls were selected for age and sex to match thepatient group. The controls were free of significant medical problems asdetermined by medical history and physical examination.

Ten ml of blood was collected by venipuncture, within one minute oftourniquet application, using Vacutainer tubes containing 143 USP unitsof heparin. The control samples were collected at the same time as thosefrom the patients and all samples were collected during afternoon hours.Plasma was isolated by centrifugation of the whole blood at 2000× g forten minutes followed by the separation of the plasma from the packed redand white cells by pipetting. The plasma was frozen at -20° C. prior toshipment (in dry ice) to the laboratory for analysis. The samples werestored at -70° C. until electrophoresis, which was performed withinthree months of venipuncture.

Gel electrophoresis. Plasma samples were thawed and 20 ul of each samplewere added to 20 ul of denaturing solution, containing 10% w/v SDS and2.3% DTT w/v? The samples were then heated to 95° C. for 4 minutesfollowed by cooling to room temperature. Then 96 ul of electrophoresissolution, containing 0.1 g DTT, 0.4 g CHAPS, 5.4 g urea, 0.5 ml pH3.5-10 ampholytes and 6.5 ml deionized water were added to each sample.The samples were mixed on a Vortex mixer and 10 ul of each processedsample (containing 1.47 ul plasma) were added to the first dimensionisoelectric focusing (IEF) gels. Isoelectric focusing was performed in3% (w/v) acrylamide gels with 4% w/v ampholytes (containing a 1:1mixture of pH 3.5-10 and pH 5-7 ampholytes) and crosslinked with 0.03%diacryloylpiperazine, i.e., 3% T/1% C. Electrophoresis was performed for18,000 volt hours, beginning with 1000 volts for 17 hours followed by2000 volts for 30 minutes.

The second dimension, wherein proteins are separated by mass, wasperformed with 160 cm×200 cm×1.5 mm slab gels using a Bio-Rad Protean IIchamber. These gels were formed with 12.2% acrylamide (w/v), 0.2MTRIS-HCl (pH 8.8), 0.7% sodium thiosulfate (w/v), 0.3%diacryloylpiperazine (w/v), 0.5% 1,4-dimethylpiperazine (v/v), and 0.07%ammonium persulfate (w/v). Electrophoresis was performed at 7° C. with aconstant current of 40 mA per gel until a dye front reached at or nearthe bottom of the gel.

Silver staining. At the end of the run, the gels were removed from theglass plates and washed for 5 minutes in water (no protein loss isdetected during this period). The gels were then soaked in a solution ofethanol/acetic acid/deionized water (40/10/50) for one hour on anorbital shaker at 36 rpm. This solution was then replaced with asolution of ethanol/acetic acid/deionized water (5/5/90) and the gelswere soaked for at least 3 hours. The gels were washed with deionizedwater for 5 minutes and soaked in 10% gluteraldehyde solution for 30minutes. Extensive washes with deionized water were performed toentirely remove the gluteraldehyde 3×10 and 4×30 minutes). [Colddeionized water (<15° C.) removes gluteraldehyde more efficiently.] Thegels were then stained for 10 minutes in an ammoniacal silver nitratesolution (6 g of silver nitrate dissolved in 30 ml of deionized water),which is slowly mixed into a solution containing 160 ml of water, 10 mlof concentrated ammonium hydroxide, and 1.5 ml of sodium hydroxide, 10mol/liter; this solution is then diluted with deionized water to a finalvolume of 750 ml) (solution H). The temperature of solution H was 20° C.After staining, the gels were washed with deionized water for 5minutes×3. The image was then developed in a citric acid andformaldehyde solution (0.1 g citric acid and 1 ml formaldehyde in 1liter of deionized water) (solution V) until a slight background stainappeared. (The optimum temperature of solution V is 15°-18° C.) Thedevelopment process was stopped with an acetic acid/deionized watersolution (5/95) for at least 15 minutes. Stained gels were stored in aglycerol/ethanol/deionized water solution (7/10/83). [This stainingmethod is described in Hochstrasser et al., Analytical Biochemistry,173, pp. 424-435 (1988).]

Gel analysis. In order to quantitate proteins, gels were scanned with anEikonics Series 78/99 digital scanner and the gel images created thuswere analyzed using PDQUEST software (Protein Database, Inc., HuntingtonStation, N.Y.) on a SUN 4-260 minicomputer. Gel images were normalizedfor protein loading and staining variation using the average log-rationormalization procedure of the PDQUEST software. Thirteen hundredproteins were analyzed on each gel and the proteins were matched andcompared quantitatively. This analysis was performed by using thePDQUEST software aided by visual examination and operator interventionin gel areas containing complex spot patterns. All proteins whichincreased or decreased in concentration by three-fold or more, and werefound to be statistically significant (by way of the Student t test orthe log Student t test) were considered to be spots of interest.

FIG. 1 represents a stained gel of a plasma sample from a controlsubject. Marker proteins are designated by the prefix M, and Table Ilists the marker plasma proteins landmarked on the gel. Proteins whichwere found to be increased or decreased in patients with CLP are listedin Tables II and III, respectively, and are indicated by number on thegel shown in FIG. 1. Those proteins which are absent in patients' plasmaare illustrated by open circles on the control gel in FIG. 1. Acomputer-generated master map of the proteins analyzed in this Exampleis represented FIG. 2.

                  TABLE I                                                         ______________________________________                                        Landmarked Plasma Proteins                                                    Identification No.                                                                              Protein                                                     ______________________________________                                        M1                beta-Haptoglobins                                           M2                alpha.sub.1 -Antitrypsins                                   M3                Albumin                                                     M4                IgG heavy chains                                            M5                IgG light chains                                            M6                Apo A-1 lipoproteins                                        ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Plasma Proteins Increased With CLP                                            Fold                                                                                                               In-                                                                           crease                                   Pro- Controls (N = 9)                                                                              Patients (N = 10)                                                                             or                                       tein Mean            Fre-  Mean        Fre-  De-                              ID#  Conc.*  S.E.M.  quency                                                                              Conc.*                                                                              S.E.M.                                                                              quency                                                                              crease                           ______________________________________                                        4210 0.0     0.0     0     51.5  24.2  5     >51.5                            1204 0.0     0.0     0     46.5  12.3  8     >44.5                            1318 0.0     0.0     0     42.5  15.3  7     >42.5                            1324 0.0     0.0     0     38.3  14.7  6     >38.3                            1702 0.0     0.0     0     31.2  13.9  4     >31.2                            4821 0.0     0.0     0     31.2  16.6  4     >31.2                            0508 0.0     0.0     0     26.4  9.2   5     >26.4                            3211 0.0     0.0     0     25.5  9.7   5     >25.5                            4109 0.0     0.0     0     21.9  10.1  4     >21.9                            5524 0.0     0.0     0     20.2  10.5  4     >20.2                            7622 0.0     0.0     0     19.8  8.2   4     >19.8                            6331 0.0     0.0     0     13.0  5.4   4     >13.0                            8322 0.0     0.0     0     10.1  4.5   4     >10.1                            3151 1.6     1.6     1     46.8  15.2  7     29.3                             1323 2.0     2.0     1     50.0  18.2  6     25.0                             5240 3.4     3.4     1     69.2  30.7  6     20.3                             2105 2.0     2.0     1     31.5  11.8  6     15.8                             1316 4.9     4.9     1     75.2  18.1  9     15.3                             6104 7.4     4.7     2     94.5  41.2  6     12.8                             7109 18.5    12.3    3     131.2 42.9  6     7.1                              3606 63.7    30.5    4     402.8 130.0 8     6.3                              1305 17.3    7.4     5     103.8 28.0  9     6.0                              3146 3.7     3.7     1     21.0  4.8   7     5.7                              7114 23.3    15.5    2     118.2 38.5  7     5.1                              7448 67.7    46.3    3     283.4 79.4  9     4.2                              3203 4.2     4.2     1     17.5  8.2   6     4.2                              0609 37.3    19.1    3     148.3 46.5  8     4.0                              0604 62.9    29.0    4     236.0 61.5  9     3.8                              7711 15.4    15.4    1     49.4  17.3  6     3.2                              5615 89.1    89.1    1     226.8 64.9  8     2.5                              ______________________________________                                         *Concentrations are in arbitrary density units.                               p is less than or equal to 0.05                                          

                  TABLE III                                                       ______________________________________                                        Plasma Proteins Decreased With CLP                                            Fold                                                                                                               In-                                                                           crease                                   Pro- Controls (N = 9)                                                                              Patients (N = 10)                                                                             or                                       tein Mean            Fre-  Mean        Fre-  De-                              ID#  Conc.*  S.E.M.  quency                                                                              Conc.*                                                                              S.E.M.                                                                              quency                                                                              crease                           ______________________________________                                        4737 224.0   152.1   5     0.0   0.0   0     >224.0                           4624 112.5   52.0    4     0.0   0.0   0     >112.5                           7212 78.1    28.1    6     0.0   0.0   0     >78.1                            7309 13.0    5.8     4     0.0   0.0   0     >13.0                            7214 11.7    6.0     3     0.0   0.0   0     >11.7                            2806 11.6    4.7     4     0.0   0.0   0     >11.6                            5013 10.8    5.6     3     0.0   0.0   0     >10.8                            4614 12.5    4.4     6     2.3   1.6   2     5.4                              3155 76.0    28.5    7     16.5  9.1   4     4.6                              8309 53.8    11.0    9     13.1  4.7   5     4.1                              8418 744.1   185.6   8     191.1 108.6 5     3.9                              7455 95.6    26.8    7     25.5  10.9  4     3.7                              5411 79.4    23.4    7     25.7  13.2  3     3.1                              7515 8.2     2.6     6     3.2   1.9   3     2.6                              ______________________________________                                         *Concentrations are in arbitrary density units.                               p is less than or equal to 0.05                                          

EXAMPLE 2

In a further, expanded study ("second study"), 36 patients with chroniclow back pain (two cases having lumbar and thoracic pain--cases 14 and32) and controls, were subjected to the methods set forth in Example 1.This protocol was approved by the Institutional Review Board of St.Luke's Hospital in Houston, Tex. All CLP patients (n=36), selected fromone of the present inventors' (BMC) clinical practice, all normalvolunteers (n=28), and all patients with conditions other than CLP(n=34) (see further Example 3) signed an approved consent form and wererequested, prior to blood sampling, to remain drug-free for at least oneweek. The CLP patients were diagnosed by clinically correlating theinformation obtained from their medical history, physical examination,x-ray examination, selected imaging techniques (MRI and/or CT scanning)and invasive techniques when indicated (myelograms and/or discograms).Healthy controls were questioned regarding and CLP symptoms and wereeliminated if they had any history of CLP. Patients with the otherconditions (see further Example 3) were diagnosed using standard historyand physical examinations and the indicated laboratory, x-ray andimaging techniques.

Venapuncture from the anticubital fossa was performed and each 5 mls. ofblood was collected into a Vacutainer tube containing 143 USP units ofsodium heparin. It was centrifuged at 783×G for ten minutes at roomtemperature and the plasma was removed and placed into a plastic tubeand frozen at -20° C. These samples were shipped overnight to theelectrophoresis facility and remained frozen at -70° C. until used. Theplasma samples were subjected to 2-D gel electrophoresis and staining asin Example 1.

In an effort to identify new spots, the patient gels were compared tocontrol gels visually without the aid of computer analysis, as well asalso using the computer analysis. It was observed that one particularprotein(s) spot appeared in patients and was virtually absent incontrols. It is postulated that the computer did not identify this spotbecause it was incorporating it into another, very closely located spoton the gel. This new spot has been designated 1bp13-14.719 (or spot719). Its presence is indicated on FIGS. 12-14 and 18-20, and thecorresponding absence of the spot on the control gels is indicated by anopen circle in FIGS. 15-17. In FIGS. 21-23 (control gels), one can seethe corresponding absence of the spot which is visible in FIGS. 18-20.

A summary of the data obtained in this study is shown in Tables IV andV. In gels where spot 719 was barely or not visible, they were scored asnegative (-). In gels which clearly contained the spot, they were scoredas positive (+). The data in Table V were obtained from three separatestudies: two independent blinded studies and a third nonblinded study.The studies were "blind" to the extent that the investigator performingthe analyses was unaware which gels were of back pain samples and whichwere controls. As can be seen from Tables IV and V, the accuracy inpredicting which samples belonged to which group is remarkably high(97.22% overall). Moreover, this new spot allows for visualidentification without the need for computer-assisted analysis, therebysimplifying the diagnostic procedure. In order to quantify the increasein spot 719, a portion of the gels were subjected to computer-assistedanalysis using the ELSIE software (developed by Mark Miller and ArthurOlson at the National Cancer Institute, U.S.A.), the results of whichare shown in FIGS. 25a and b.

                                      TABLE IV                                    __________________________________________________________________________    Back Pain Study of Example 2                                                  Pt.                                                                              Age Sex WC  Atty                                                                              Surg                                                                              719 Diagnosis      Med                                 __________________________________________________________________________    1. 31  M   +   +   1   +   HNP L5-S1.     0                                   2. 35  M   +   0   1   +   HNP L4-5, L5-S1; Scar.                                                                       0                                   3. 47  F   0   0   1   +   Spondylosis, HNP L4-5.                                                                       0                                   4. 56  F   0   0   1   +   Pseudoarthrosis L4-5.                                                                        T13; R                              5. 44  M   +   +   2   -   Donor Site Pain.                                                                             0                                   6. 43  M   +   +   5   +   Pseudoarthosis L4-S1;                                                                        0                                                              Stenosis L3-4, L4-5; Scar                          7. 24  M   +   +   0   +   HNP L4-5, L5-S1.                                                                             T13                                 8. 49  M   0   0   2   +   Stenosis L2-3; VIC                                                            Pseudoarthrosis, L4-5.                             9. 43  M   +   +   5   +   Deg. Spondylolisthesis                                                                       0                                                              L3-4; Pseudoarth. L4-S1.                           10.                                                                              37  M   +   +   1   +   HNP, Spondylosis,                                                                            0                                                              Stenosis L4-5.                                        47  M   0   0   1   +   HNP L4-5, L5-S1.                                                                             0                                      26  M   +   +   2   +   Spondylolysthesis L5-S1;                                                                     0                                                              Pseudoarthrosis L5-S1.                                30  M   +   +   2   +   Recurrent HNP L4-5.                                                                          0                                      60  F   0   0   0   +   Osteoporosis,  0                                                              Osteoarthritis.                                       50  F   0   0   0   +   Deg. Spondylolisthesis                                                                       0                                                              L4-5; DDD. L4-5; L5-S1.                               63  M   +   0   0   +   HNP L4-5; L5-S1.                                                                             0                                      44  M   +   0   4   +   DDD, HNP,      0                                                              Pseudoarthrosis L4-5.                                 48  M   +   +   2   +   DDD, HNP, Spondylosis,                                                                       0                                                              Pseudoarthrosis, L4-5.                                26  F   +   0   0   +   HNP L5-S1; DDD L4-5.                                                                         0                                   20.                                                                              42  F   +   +   2   +   Lumbar Stenosis;                                                                             0                                                              Pseudoarthrosis, L4-5.                                49  M   +   +   5   +   HNP L5-S1; Arachnoiditis.                                                                    0                                      27  F   +   0   0   +   HNP L4-5.      0                                      34  M   +   +   0   +   HNP L4-5.      0                                      44  M   0   0   1   +   HNP L4-5.      0                                      44  F   +   +   1   +   HNP L4-5.      VIC                                    26  M   +   +   0   -   Spondylosis L5-S1.                                                                           0                                      52  F   +   0   3   +   DDD, Spondylosis L3-4;                                                                       0                                                              HNP L4-5.                                             48  M   +   +   1   +   HNP L3-4; Buldge L4-5.                                                                       0                                      29  M   +   +   3   +   Lumbar Stenosis L5-S1;                                                                       0                                                              Sacralization L-5                                  30.                                                                              38  F   +   +   2   +   HNP L4-5, L5-S1;                                                                             NAP.                                                           DDD L4-5; Stenosis L5-S1.                                                                    ROB.                                   52  M   +   0   2   +   HNP, Spondylosis L4-5.                                                                       0                                      59  F   0   0   0   +   Scoliosis; Spondylosis.                                                                      0                                      31  M   +   +   6   +   HNP L5-S1; Facet damage                                                                      0                                                              L5; Arachnoiditis.                                    42  M   +   +   3   +   HNP, Instability L3-4;                                                                       0                                                              Stenosis L4-5.                                        31  M   +   +   2   +   HNP, facet damage L5-S1.                                                                     5 0                                    34  M   +   +   1   +   Spondylolisthesis L5-S1.                                                                     0                                   __________________________________________________________________________

Table IV. Clinical data from 36 CLP patients in a study which included28 controls: WC=workman's compensation case; Atty=attorney involvement;Surg=number of back operations at the time of venapuncture;719=increased (+) or decrease (-) in the plasma level of theapolipoprotein E variant scored as described in Methods; HNP=herniatednucleus pulposis; DDD=degenerative disc disease; Med=drugs;NAP=Naprosyn; ROB=Robaxin; VIC.=Vicodin; T#3=Tylenol #3.

                  TABLE V                                                         ______________________________________                                        Back Pain Study of Example 2                                                                                    Patients                                                                      and                                         Subjects  CLP Patients                                                                              Controls    Controls                                    ______________________________________                                        Apo E increase                                                                          34+     2-      0+    28-   62/64                                   Correlation                                                                             34/36       28/28       62/64                                       Test      Sensitivity Specificity Efficiency                                  Percentage                                                                              94.44%      100.00%     97.22%                                      ______________________________________                                         Table V. Statistical analysis of CLP data: Results of the plasma studies      of 36 CLP patients and 28 normal controls. The efficiency was calculated      as = (sensitivity + specificity)/2.                                      

EXAMPLE 3

Five additional groups of patients' plasma were analyzed with respect to1bp13-14.719. These groups consisted of:

A. Three patients who were relieved of their CLP through surgery and twounoperated, asymptomatic individuals who had biomechanical abnormalitiesthat often produced chronic lower back pain. None of these patientsdisplayed an increase in the apolipoprotein E variant (spot 719)associated with chronic lower back pain (see Table VI).

B. In order to determine the significance of the location of the pain inthe spinal column in inducing the presence of the apolipoprotein Evariant from patients with CLP, plasma samples were analyzed from fourindividuals with chronic cervical pain (CCP). All four patients with CCPdemonstrated the apolipoprotein E variant (spot 719) (see Table VII).

C. To determine if normal nociception without nerve damage induced anincrease in the apolipoprotein E variant (spot 719), the plasma proteinsof 11 patients with painful orthopedic injuries without nerve damagewere analyzed. Only one of these patients, who may have sustained nervedamage, displayed an increase in the apolipoprotein E variant (see TableVIII).

D. Nerve damage is thought to be associated with CLP. To determinewhether other peripheral nerve damage causes the induction ofapolipoprotein E variant (spot 719) and plasma, which is associated withCLP, the plasma samples of six individuals with various peripheral nerveproblems were examined. Five of the six displayed an increase in theapolipoprotein E variant. The remaining patient had a painless radialnerve palsy. (See Table IX).

E. Local inflammatory responses (which often include edema anddemyelination) of the nerve root have been demonstrated with chroniclower back pain. To determine if the increase in apolipoprotein E (spot719) was associated with known inflammatory conditions, the plasma ofseven patients with chronic systemic inflammatory changes (lupuserythematosus, rheumatoid arthritis, and Crohn's disease) was studied.Three patients with Crohn's disease and two with lupus displayed anincrease in the apolipoprotein E variant (spot 719), while the two withrheumatoid arthritis did not. (See Table X).

The above samples were subjected to the methods and analyses of Example2, wherein the presence or absence of 1bp13-14.719 was determined.

These studies were undertaken to answer the questions previously posedin this specification. In particular, (1) is pain required or is only abiomechanical change required to increase the plasma level of theapolipoprotein E variant? In Group A, five asymptomatic patients withbiomechanical changes were tested: two with spondylolisthesis, two withpost-operative discectomies, and one with a post-operative discectomyand fusion. None exhibited an increase in the apolipoprotein E variant.This study indicated the association of the protein (spot 719) withpain, and also strongly suggested that the protein difference was notdue to painless surgical scarring (see Table VI).

(2) Is the induction of the apolipoprotein E variant (spot 719) from thespine limited only to chronic pain in the lumbar region? To determine ifthe increase in the plasma level of apolipoprotein E variant is specificfor pain in the lumbar region, we examined the plasma of four patientswith CCP (Group B). All four demonstrated a significant increase in thisprotein. This study indicated that pain associated with nerve damageanywhere in the spinal column may be associated with increased plasmaconcentrations of this protein.

(3) Is the apolipoprotein E variant increased in all patients with acuteor chronic pain? For this study, the area of nociception was extended tothe periphery and 11 patients were selected with various painfulconditions without nerve damage (Group C). These conditions ranged froma fractured ulna to a ligamentous shoulder injury. Ten were negative foran increase (90.9%) and one was positive for an increase in theapolipoprotein E variant (see Table VIII). This one patient had amultitude of serious orthopedic injuries (with probable nerve damage),and it could not be determined which of them was responsible for thepositive result. This study suggested that peripheral pain without nervedamage does not produce the protein (spot 719).

(4) Is an increase in the plasma apolipoprotein E variant found inpatients with painful conditions associated with peripheral nervedamage? An increase in this protein was found in individuals withchronic pain associated with the vertebral column, but was not found inindividuals with peripheral pain who did not have evidence of nervedamage. This suggested that the protein variant found in the plasma ofchronic spinal column pain patients was associated with chronic nervedamage, but was not associated with normal nociception with no nervedamage. On the other hand, it was postulated that peripheral pain due tonerve damage would be associated with an increase in the plasma level ofthe apolipoprotein E variant (spot 719). To test this hypothesis, theplasma from six patients with peripheral nerve damage (Group D) wastested. (See Table IX). Five of six were positive for an increase in theplasma concentration of the apolipoprotein E variant. The patient withan adventitious bursa was later diagnosed with cervical spondylosis andoperated with relief of her symptoms. The one patient who displayed noincrease in this protein had a painless radial nerve palsy. Sinceapolipoprotein E is known to be associated with nerve regeneration, itis possible that there was no physiological stimulus to repair thenerve. The incidence of spontaneous regeneration of the radial nervefollowing fractures of the humerus has been reported variously to be70-92% with neuropraxia or axonotomesis, but 0% with neuronotomesis. Theabsence of an increase in the plasma level of apolipoprotein E variantand the absence of regeneration correlated with the clinical picture.From this study and those above, it is reasonable to suggest that nervedamage and regeneration are necessary to produce an increase in theplasma level of the apolipoprotein E variant (spot 719). Thus, theabsence, or substantial absence, of spot 719 in a patient diagnosed byconventional methods to have peripheral nerve damage (for example, aradial nerve palsy), would indicate that there has been a lack of nerveregeneration. This would guide the clinician to treat the patient withtherapy known to stimulate nerve regeneration. This would be especiallyhelpful to a clinician watching the clinical course of a brachial plexusinjury, a radial nerve palsy, a peroneal palsy, a peripheral neuropathy,a causalgia, etc.

(5) Is an increase in plasma apolipoprotein E variant (spot 719) relatedto inflammation? In addition to stimulating regeneration, nerve damagehas been noted to stimulate a local, neural inflammatory response. It ispossible that mediators of inflammation are necessary for nerveregeneration, because apolipoprotein E is known to be involved in bothresponses. To answer this, a group of patients with diseases known tostimulate chronic inflammation (Crohn's disease, lupus erythematosus andrheumatoid arthritis) was studied (Group E). Three patients with Crohn'sdisease and two with lupus were positive; but the two with rheumatoidarthritis were negative (see Table X). From this study, it is possibleto conclude that the increase in the plasma concentration of thisapolipoprotein E variant may be associated with chronic inflammatorydiseases, which can produce false positives with respect to thediagnosis of peripheral nerve damage, and in particular, CLP and CCP.

It is known in the literature from biochemical studies of crushedsciatic nerves in rats, rabbits and primates that apolipoprotein E wasessential for nerve repair in mammals, and that its local concentrationmay be increased 250-fold. There is evidence that apolipoprotein Eenters the nerve to a limited degree from the plasma, but it is producedin massive quantities by the resident macrophages and endothelial cellswithin the nerve, as well as monocyte-derived macrophages which enterthe nerve in response to denervation. It is thought that apolipoproteinE may be involved in the redistribution of lipid (involving macrophagesand Schwann cells), including the cholesterol released duringdegeneration to the regenerating axons. Apolipoprotein D, apolipoproteinA-I and AIV are also thought to be associated with this lipid transfer.Although these other apolipoproteins have been observed in the presentstudies, it is significant to note that these proteins do not appear tobe useful as diagnostic markers of peripheral nerve damage. Forinstance, apo-D is reported in the literature to be locally increased(around the nerve tissue) 500-fold, but a corresponding increase in theplasma is not seen (see FIGS. 25a and b). The experimental data andhuman data in the prior art indicate that nerve pressure or tension canproduce intraneural vascular changes, which ultimately result indegeneration which stimulates regeneration. It was postulated in theprior art that these perturbations produced a loss of nerve functionand/or hyperexcitability with pain from ectopic generation. The increasein apolipoprotein E, which has been observed in animal nerve injurystudies, is consistent with the increased plasma levels withapolipoprotein E variant observed in the plasma of patients with nerveinjury in this study. This protein alteration may be part of a normalphysiological response to nerve damage, and its presence can serve as auseful marker in the disorders associated with nerve damage, inparticular in the diagnosis and management of patients with CLP and CCP.

                  TABLE VI                                                        ______________________________________                                        GROUP A                                                                       Patient  Age    Sex      Diagnosis  Spot 719                                  ______________________________________                                        1        67     M        Spondylolisthesis                                                                        -                                                                  L4-5                                                 2        45     M        Spondylolisthesis                                                                        -                                                                  L5-S1                                                3        49     M        Lam., Disectomy,                                                                         -                                                                  HNP L5-S1                                            4        59     M        Lam., Disectomy,                                                                         -                                                                  Fusion, L5-S1                                        5        44     M        Lam., Disectomy,                                                                         -                                                                  L5-S1                                                ______________________________________                                    

                  TABLE VII                                                       ______________________________________                                        GROUP B                                                                       Patient                                                                              Age    Sex     Diagnosis      Spot 719                                 ______________________________________                                        1      35     F       Cervical Spondylosis                                                                         +                                        2      39     F       Cervical Spondylosis                                                                         +                                        3      38     F       Cerv. Spondy.; Lam.,                                                                         +                                                              fusion C5-6                                             4      41     M       Cervical Spondy.; Detach.                                                                    +                                                              Deltoid.                                                ______________________________________                                    

                  TABLE VIII                                                      ______________________________________                                        GROUP C                                                                       Patient                                                                             Age     Sex    Diagnosis       Spot 719                                 ______________________________________                                        1     68      F      Lumbar Spondylosis; THR.                                                                      -                                        2     59      M      Failed THR.     -                                        3     22      M      Early Aseptic Necrosis,                                                                       -                                                             hip.                                                     4     34      M      Ligamentous Tear,                                                                             -                                                             Shoulder                                                 5     15      M      Right Knee MCL Sprain                                                                         -                                        6     34      M      Asep. Nec. L Hip; Discl.                                                                      -                                                             SC Jt.                                                   7     47      F      Shoulder Pain.  -                                        8     34      F      Fractured Right Ulna,                                                                         -                                        9     40      M      Asept. Necrosis, Bilat.                                                                       -                                                             Hips                                                     10    29      F      Knee Pain: Synovitis,                                                                         -                                                             tear MM.                                                 11    58      M      Failed THR; Multi. Comp.                                                                      +                                                             Fracts.                                                  ______________________________________                                    

                  TABLE IX                                                        ______________________________________                                        GROUP D                                                                       Patient                                                                              Age    Sex     Diagnosis      Spot 719                                 ______________________________________                                        1      24     M       Radial nerve palsy; frct.                                                                    -                                                              humerus                                                 2      42     F       Cubital Tunnel Syndrome                                                                      +                                        3      47     M       Morton's Neuroma                                                                             +                                        4      33     M       Reflex Sympathetic                                                                           +                                                              Dystrophy                                               5      47     M       Left knee; Failed fixator                                                                    +                                        6      46     F       Adventitious Bursa, Left                                                                     +                                                              scapula                                                 ______________________________________                                    

                  TABLE X                                                         ______________________________________                                        GROUP E                                                                       Patient                                                                              Age     Sex     Diagnosis     Spot 719                                 ______________________________________                                        1      32      F       Crohn's Disease                                                                             +                                        2      27      F       Crohn's Disease                                                                             +                                        3      36      F       Crohn's Disease                                                                             +                                        4      33      F       Crohn's Disease                                                                             +                                        5      33      F       Lupus.        +                                        6      60      F       Rheumatoid Arthritis.                                                                       -                                        7      76      M       Rheumatoid Arthritis.                                                                       -                                        ______________________________________                                    

EXAMPLE 4

Immunoblot analysis was conducted for spot 719. Proteins separated byone and two dimensional gel electrophoresis electro-blotted on PVDFmembranes (Immobilon-P, Millipore), prepared according to themanufacturer's instructions, for 40 minutes at 0.8 amps using atransblot (BioRad) semi-dry electroblotter. The blots were then driedand placed in methanol followed by blocking overnight with PBScontaining bovine serumalbumin. ECL (Amersham) detection was performedaccording to the manufacturer's instructions using 1:1000 dilution ofmonoclonal anti-apo-E (Chemicon) for two hours at room temperature and1:25000 antimouse immunoglobulin (Pierce) for 30 minutes. Spot 719 waspositive for anti-apo-E reactivity (FIG. 26). Other modified forms ofApo-E were also detected. These latter spot densities were analyzed bycomputer-assisted densitometry for quantitative variations correlatingwith chronic lower back pain, and there were none (FIGS. 25a and 25b).

EXAMPLE 5

Amino acid N-terminal microsequence of amido black-stainedelectroblotted protein was performed. Automated peptide microsequencingwas performed by dansyl-Edman degradation as described by B. S. Hartley(1970), Strategy and Tactics in Protein Chemistry, Biochem. J., Vol.119, pp. 805-822, with modifications described by John M. Walker (1984)Proteins, Vol. 1, The Humana Press, pp. 221-242. In order to determinethe preparative ability of the 2-D gel, the maximum plasma proteincapacity for resolving spots 719 was determined by titering the sampleload. It was determined that the optimal load for 2 D resolution andvisualization with amido black of spot 719 was 60 microliters of sample(three times the analytical load). Spot 719 was excised from five amidoblack-stained preparative 2-D gel electroblots. N-terminal microsequenceanalysis was performed on spot 719 and was determined to be apo-E (FIG.27). N-terminal sequence analysis determined 100% homology with theknown sequence for apo-E.

EXAMPLE 6

To determine whether there is a correlation between protein spot densityand the severity of pain, the densities of the various proteins ofinterest in the initial study were compared to the degree of lower backdisability, as scored by historical as well as physical and radiologicaldeterminations.

A short, abbreviated scoring system (abbreviated from a scale of 144possible factors) was devised which is similar to the Waddell approach[Waddell and Main, "Assessment of severity in low-back disorders", SpineVol 9 pp. 204-208 (1984); Waddell, Main, Morris, Paola and Gray, 1984;Waddell et al., 1980]. The physical signs which Waddell et al selectedas being significant were: degree of lumbar flexion; straight legraising; root compression signs; and previous lumbar surgeries. The sixclinical signs used in the abbreviated scale were similar andconstituted the clinical objective scale (COS):

1. Scar from previous back surgery. All back surgery creates permanentscarring with permanent changes, no matter how subtle. This contributesto the presence of minute to major back pain. Each surgery scored 2.

2. True spasm which the patient cannot control (the most significantsign of all). This is not a limitation of motion which the patient cancontrol, but true, uncontrolled spasm which the physician can recognize.Although sometimes painless in a condition such as burned out ankylosingspondylitis, it otherwise invariably signals severe pain. The physiciancan easily recognize this exception. This finding scored 4.

3. Straight leg raising (SLR) (right and left). This must bedifferentiated from hamstring spasm and a functional SLR. Hamstringspasm or tightness produces pain locally in the thigh, not in the backwith SLR. Functional SLR is easily recognized by having the patient siton the edge of the table and casually extend the knee. If the patientdoes not complain of back pain, there is no positive SLR. Inknowledgeable hands, this is an objective finding. Each SLR scored 2.

4. Knee or ankle reflex change (right and left). These are objectivefindings in all cases. It is possible for them to be present withoutpain because of old trauma or surgery. Some reflex changes areassociated with weakness or atrophy, but because these findings arefrequently not recorded, it is not practical to score the latter. Eachreflex change scored 1.

Table XI summarizes the COS.

                  TABLE XI                                                        ______________________________________                                        Summary of COS                                                                FACTOR                VALUE                                                   ______________________________________                                        Each back incision (scar)                                                                           2                                                       Spasm                 4                                                       Right Straight Leg Raising (RSLR)                                                                   2                                                       Left Straight Leg Raising (LSLR)                                                                    2                                                       Right Reflex Change (RRC)                                                                           1                                                       Left Reflex Change (LRC)                                                                            1                                                       ______________________________________                                    

The abbreviated scoring of the patients of this study are seen in TableXII, below. The numbers indicated next to the gel are arbitrarilyassigned patient numbers.

                  TABLE XII                                                       ______________________________________                                        Scores of COS of Patients.                                                                                                 TO-                              Patient                                                                             SCAR    SPASM    RSLR  LSLR  RRC  LRC  TAL                              ______________________________________                                        gel 1 2       4              2               8                                gel 3                        2               2                                gel 5                                        0                                gel 7 2                2                     4                                gel 9                                        0                                gel 11                 2     2     1         5                                gel 13                                                                              2                      2               4                                gel 15                                                                              2       4        2     2          1    11                               gel 17                                       0                                gel 19                                                                              2                      2          1    5                                ______________________________________                                    

By comparing the above COS scores to the amount of protein 1318 in thegels, it was found that the protein designated 1318 displayed acorrelation with pain severity. This is represented in FIG. 5.

The same comparison was made with proteins 1316, 1204, 1305, 3203 and3211. Also a comparison was made with the average of proteins 1318 and1316. These results are shown in FIGS. 6-11.

Using similar methods, a correlation may also be made with spot 719 andany of the conditions which result from peripheral nerve damage.

EXAMPLE 7

To examine the effectiveness of a course of treatment for conditionsresulting from peripheral nerve damage, a blood sample would be obtainedfrom a patient prior to the treatment. A two-dimensional gel orimmunoassay would be run, stained and analyzed for levels of one or moreof the proteins listed in Tables II, III,IV and V to obtain a baseline.After instituting treatment, one or more blood samples are taken fromthe patient and analyzed for levels of the same protein or proteinswhich were initially analyzed. A proportional increase or decrease tonormal levels (depending on whether the protein analyzed is one which isfound to increase or decrease in the patients) signifies that thetreatment is successful.

EXAMPLE 8

A. Preparing Antigens. After two-dimensional gel electrophoresis isperformed on a patient, for instance one with confirmed CLP, the gelwould be stained with Coomassie blue in order to locate a protein ofinterest. The gel is rinsed with deionized water for a few minutes,changing the water several times. The spot containing a protein is cutout of the gel with a scalpel, and placed on a piece of parafilm orplastic wrap. The edge of a paper towel is used to remove by capillaryaction any standing water. Next, the plungers from the barrels of two 5cc syringes are removed, and the gel piece is placed into one of thebarrels. The plunger is then replaced and the syringe outlet ispositioned in the barrel of the second syringe. Using rapid, firmpressure on the plunger, the gel is pushed into the barrel of the secondsyringe. This process is repeated several times back and forth betweenthe two syringes. Then, 21-gauge needles are placed onto the outlet ofthe syringes, and the process is repeated. A small amount of buffer(PBS) may be necessary to keep the small fragments passing back andforth between the syringes. The samples are now ready for injection.

B. Preparing Antisera. Antibodies are raised in rabbits immunized byinjecting the antigen preparation (above). An initial subcutaneousinjection of approximately 150 ug of one of the protein preparationswould be followed by two monthly injections of approximately 100 ug ofthe antigen. This will lead to a sufficient antibody titer for use in animmunoassay.

C. Preparing Monoclonals. Monoclonal antibodies may be preparedaccording to the method of K ohler and Milstein. This method involvesimmunizing mice with an antigen bearing one or more epitopes (i.e., oneof the lower back pain proteins). The mice develop spleen cells makinganti-epitope(s) which appear as an antibody (or antibodies) in theserum. The spleen is removed and the individual cells fused inpolyethylene glycol with constantly dividing (i.e., immortal) B-tumorcells selected for a purine enzyme deficiency and often for theirinability to secrete Ig. The resulting cells are distributed intomicro-well plates in HAT (hypoxanthine, aminopterin, thymidine) mediumwhich kills off the perfusion partners, at such a high dilution that, onaverage, each well will contain less than one hybridoma cell. Eachhybridoma being the fusion product of a single antibody-forming cell anda tumor cell will have the ability of the former to secrete a singlespecies of antibody and the immortality of the latter enabling it toproliferate continuously, clonal progeny providing an unending supply ofantibody.

D. Western Blot (Immunoblot). Proteins from the two-dimensional gels ofExamples 1 and 2 may be electrophoretically eluted, prior to staining,to 0.2 um nitrocellulose membranes. The membranes are rinsed with PBSand incubated with BSA (3% BSA (fraction V, 0.02% sodium azide in PBS).The membranes are then incubated with primary rabbit polyclonalantibodies (obtainable by the above method) at a concentration of aboutbetween 1 and 50 ug/ml in PBS. The membranes are then washed withseveral changes of PBS, followed by incubation with goat anti-rabbit IgGlabeled with horseradish peroxidase. Finally, after rinsing with PBS,the membranes are developed with 4-chloro-1-naphthol (stock solution is0.3 g chloronaphthol in 10 ml absolute ethanol; working solution is 0.1ml stock added to 10 ml of 50 mM TRIS, pH 7.6; the white precipitate isfiltered and 10 ul of 30% hydrogen peroxide is added). The reaction isstopped by rinsing with PBS. A positive result is seen if the spots ofinterest develop into a blue-black color.

In the foregoing immunoblot procedure, monoclonal antibodies may besubstituted for the primary polyclonal antisera to obtain a higherspecificity.

E. Radioimmunoassay (RIA). To perform this assay, one would use amonoclonal antibody to one of the proteins of interest (Such as thoseexemplified in Examples 1 and 2), which would be prepared according toknown methods as discussed above.

A sheet of nitrocellulose paper is cut to the size of a dot blotapparatus. The sheet is pre-wetted with water, and fitted onto theapparatus. A plasma sample from the patient is placed in the wells (30ul/well) in serial dilutions, and incubated for two hours in a humidatmosphere. The sheet is then washed with two changes of PBS. The sheetis then blocked by incubating with a solution of 3% BSA/PBS with 0.02%sodium azide for at least 2 hours. Following washing with PBS, theprimary monoclonal antibody (in a solution of 3% BSA/PBS With 0.02%sodium azide) is added at a suitable dilution and incubated for 2 hourswith agitation. Unbound antibody is washed away with PBS. An I¹²⁵-labeled goat anti-rabbit IgG (in 3% BSA/PBS with 0.02% sodium azide) isthen incubated with the sheet for about 2 hours with agitation. Unboundlabeled antibody is removed by washing four times with PBS for 5 minuteseach. The amount of bound labeled antibody is determined byautoradiographic detection. This is done by placing the sample sheet indirect contact with an X-ray film and storing this system at -70° C.with an intensifying screen. Results can be crudely quantitated byvisual examination of the exposed film and more finely quantitatively bydensitometric tracing. The relative amounts of antigen in differentsamples are determined by comparing midpoints of the titration curves.Absolute amounts of antigen can be determined by comparing these valueswith those obtained using known amounts of antigen.

F. Enzyme-linked Immunosorbent Assay (ELISA). This immunoassay isperformed as set forth above in the RIA method; however, rather than thesecondary antibody, goat anti-rabbit IgG, being labeled with I¹²⁵, it islabeled with horseradish peroxidase (HRP). In order to detect andquantify the HRP, the dot blot is developed with chloro-naphthol.4-Chloro-1-naphthol (0.3 g) is dissolved in 10 ml of absolute ethanol toprepare a stock solution. Immediately prior to developing the assay, 0.1ml of the stock is added to 10 ml of 50 mM TRIS (pH 7.6). The whiteprecipitate formed is filtered with Whatman No. 1 filter paper. 10 ul of30% H₂ O₂ is added to the solution. The chloro-naphthol solution isadded to the nitrocellulose sheet and agitated until the spots aresuitably dark (about 30 min.). The reaction is stopped by rinsing withPBS. The results can be determined as with the RIA, and quantificationperformed by visual inspection or by reflection densitometry.

EXAMPLE 9

Typical test kits for use with RIA or ELISA tests will contain:

1. A plate with absorbed rabbit Fab fragment IgG (to any of the proteinsset forth in Tables II, III, IV and V, preferably the protein of spot1bp13-14.719), or nitrocellulose sheets with the absorbed rabbit IgG.

2. Rabbit whole IgG (to the same protein as above).

3. Labeled goat anti-rabbit IgG (F_(c) portion).

B

1. Mouse monoclonal (to any of the proteins listed in Tables II, III, IVand V preferably the protein of spot 1bp13-14.719).

2. Labeled goat anti-mouse.

These kits may also contain appropriate buffers such as PBS, blockingsolution, and appropriate enzyme substrates (for ELISAs). Thesematerials may be provided with the kit or may be separately provided orprepared.

The term "plate" is used in the broad sense to include any flat surfacewhich can be employed with an RIA or ELISA.

In practice the test kit A (above) would be employed as follows:

1. Incubate the plate with the serum of the patient under test for anappropriate time and temperature (e.g., from 2-4 hours at 37° C.).

2. Wash with BSA/PBS.

3. Incubate with rabbit whole IgG and wash with buffer.

4. Incubate with labeled goat anti-rabbit IgG (F_(c) portion) and washwith the same buffer.

5. Detect the formation of a reaction product (or radioactive signal) inthe case of a positive test by any of the aforementioned procedures.

The test kit B would be employed as follows:

1. Incubate a substrate (plate, nitrocellulose paper, etc.) with anunknown sample (such as plasma) for an appropriate time and temperature.

2. Wash with BSA/PBS.

3. Incubate substrate with the mouse monoclonal and wash with buffer.

4. Incubate with goat anti-rabbit IgG and wash with same buffer.

5. Detect the formation of a reaction product (or radioactive signal) inthe case of a positive test by an appropriate procedure.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       XaaValGluGlnAlaValGluThrGluProGluXaaGluLeu                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyCysGlnAlaLysValGluGlnAlaValGluThrGluProGluPro                              151015                                                                        GluLeuArgGlnGlnThrGluTrp                                                      20                                                                            __________________________________________________________________________

We claim:
 1. An isolated and purified apolipoprotein E variantobtainable from patients with peripheral nerve damage, which, whensubject to two dimensional gel electrophoresis, migrates to spot1bp13-14.719 as shown in FIG. 24b.